Mispriming is a significant problem faced when performing primer-dependent amplification processes, such as polymerase chain reaction (PCR). Mispriming is manifest in at least four types: Type 1 mispriming, which occurs during the preparation of reaction mixtures or the execution of other enzymatic manipulations (e.g., reverse transcription in the case of one-step RT-PCR) prior to the start of amplification; Type 2 mispriming, which occurs during an amplification if cycle temperatures include any temperature significantly below the primer annealing temperature, as may occur during the performance of an asymmetric PCR amplifications, such as Linear-After-The-Exponential PCR (LATE-PCR), during which the incubation temperature may be dropped to allow probes with low melting temperatures to bind their target or at the end of an amplification reaction when the sample is cooled down and removed from the thermal cycler prior to a subsequent process such as DNA sequencing; Type 3 mispriming occurs during amplification at cycles having a temperature at or above the primer annealing temperature of the reaction; and Type 4 mispriming, which occurs in the late stages of a amplification after a high concentration of amplicon has been made. Thus, Type 1 and Type 2 mispriming occur below the primer annealing temperature of the amplification reaction, while Type 3 and 4 mispriming occur at or above the primer annealing temperature of the reaction.
One manifestation of Type 1 and Type 2 mispriming is formation of primer-dimers, which occurs when one primer hybridizes to the other primer or to itself and then undergoes extension of the 3′ end to generate a small, double-stranded amplicon. This amplicon can then undergo further amplification and/or can form an even larger oligomer. Primer-dimer formation can occur even in the absence of a target nucleic acid sequence. Among the approaches that have been applied to address Type 1 mispriming is the use of an antibody that binds to the DNA polymerase and inhibit the polymerase activity until the reaction is heated to a high temperature, such as 95° C., at which point the antibody is irreversibly denatured and can no longer bind to the polymerase.
Type 1 and Type 2 mispriming can be measured by various methods, including through the use of real-time PCR amplification monitored with fluorescent dyes that interact with double stranded DNA (e.g., SYBR Green 1). For example, for reactions containing targets, mispriming can result in threshold cycle (Ct) values that are lower than the Ct predicted for the number of starting targets and the efficiency of amplification. For reactions with no targets, mispriming can be observed as the presence of real-time amplification signals; the earlier the Ct value of these signals, the greater the incidence of mispriming. Type 1 and Type 2 mispriming can also be measured by first derivative melting curve analysis, where mispriming can be observed as the formation of melting peaks different from the melting peak of the intended amplification product, as shoulders on either side of melting peak of the intended amplification product, or as an increase in the width of the melting peak of the intended amplification product. Type 1 and Type 2 mispriming can also be detected using gel electrophoresis, in which case mispriming can be observed as bands other than the band corresponding to the predicted length of the intended specific amplification product or as higher molecular weight or lower molecular weight smears. Prevention of mispriming results in more efficient use of primers, which is manifest as an increase in the amplification of the intended product.